PROJECT SUMMARY A small population of long-lived CD4 T cells harbors replication competent virus (the latent HIV reservoir) during effective antiretroviral therapy even when viremia is undetectable; this latent HIV reservoir is invariably associated with virus rebound when treatment is stopped. The latent HIV reservoir is a major barrier to curing HIV, but multiple technical challenges limit its investigation. We developed an ultra-high throughput droplet microfluidic workflow called PCR activated cell sorting (PACS) that detects, sorts and sequences single cells containing a single copy of intracellular HIV DNA. In preliminary studies we demonstrated the ability of PACS to 1) process millions of cells at ultra-high throughput, 2) detect HIV infected cells 3) single cell sort and sequence this rare population of latently infected CD4 T cells. Based on these findings, we believe that PACS provides a unique opportunity to overcome existing technical challenges and define, for the first time, the genomic mechanisms that control the HIV latent reservoir. We hypothesize that latently infected cells have unique properties that allow them to harbor replication competent HIV genomes without producing virus. Thus, we propose to combine PACS with methods for single cell RNA-seq, genome and integration site analysis, ATAC-seq and proteomics to define the genomic mechanisms that control the HIV latent reservoir in single infected cells from people with ART suppression of the virus. Our Specific Aims are as follows: Specific Aim 1: Define the transcriptome of single cells harboring latent virus. We propose to sequence the transcriptomes of single HIV positive CD4 T cells isolated from the blood of individuals on ART, while determining in parallel the HIV provirus sequence and insertion site. These studies will define the transcriptional program of latently infected cells harboring full-length replication competent virus. Specific Aim 2: Establish the chromatin landscape of single cells harboring latent virus. We propose to analyze the chromatin profile of single HIV positive CD4 T cells in order to define the relationship between host DNA chromatin status and HIV latency. These studies will determine the role of chromatin status in the control of HIV latency and identify regulators of the transcriptional program of latently infected CD4 T cells. Specific Aim 3: Identify surface markers of latently infected CD4 T cells. We propose to isolate HIV latently infected CD4 T cells using PACS and analyze their surface proteome using barcoded antibody sequencing to identify surface marker combinations that best define the HIV latent reservoir. Upon completion, these studies will define novel surface marker combinations to identify latently infected CD4 T cells. IN SUMMARY, these studies use a multi-omics approach based on our combined expertise in the genomic regulation of immune cells, HIV biology, and microfluidics to define the mechanisms that control HIV latency. Hence, these studies will guide the development of novel therapeutic interventions, while providing novel tools for the monitoring of the latent HIV reservoir in infected individuals undergoing ART.